Download MICROPATTERNED CELL CO-CULTURES USING LAYER

THREE-DIMENSIONAL GROWTH AND FUNCTION OF NEURAL
TISSUE IN DEGRADABLE POLYETHYLENE GLYCOL HYDROGELS
M.J. Mahoney, K.S. Anseth
Department of Chemical and Biological Engineering and the Howard Hughes Medical Institute
University of Colorado,
Campus Box 424, Boulder, CO 80309, USA
MARK HWANG
POLYMER SCAFFOLDS - BACKGROUND
Uses:
- Model tissue ECM environment in vitro
- Observation platform for:
* cell-cell interaction
* cell-ECM interaction
- Testing platform for drug delivery
* pre/post –encapsulation
- Tissue replacement/grafting therapy
Key Factors:
- Mesh size
- Scaffold chemistry
POLYMER SCAFFOLDS - CENTRAL NERVOUS SYSTEM
Grafting as disease treatment limited by:
graft survival
 (viability)
integration
 (retain functionality)
Graft research focus for CNS disease
Scaffolds support neural cell
- growth
- differentiation
- function
Cell line chosen for this study:
undifferentiated (embryonic) murine neural precursor cells (NPC)
STUDY GOALS
Assess effect of 1) degradable hydrogel
on
2) neural precursor cell (NPC) viability +
3) NPC differentiation +
4) 3D tissue morphology +
given
5) mesh size that changes with time
RATIONALE
NPCs merits
- in vitro expansion before transplantation  unlimited NPC source
- from previous studies
successful transplantation into adult rats
adequate chemical microenvironment in adult CNS ECM
3D v. 2D scaffold
- directly implant 3D scaffold
- cells must be dislodged from 2D substrate
shear forces
PEG hydrogel
- non-immunogenic
- tolerated in CNS
- degradable
- protein scaffolds (e.g. collagen) hard to control
MATERIALS AND METHODS: BIG PICTURE
Overall Goals
1) Construct gel  determine degradation with mechanical tests
Does hydrogel restrict cell growth?
Does hydrogel affect viability?
Incubate cells in gel
2) Cell imaging
Stain for in gel viability
Stain for in gel bioactivity (monitor calcium level)
3) Biochemical analysis after gel/cell lysed
DNA levels
ATP levels
MATERIALS AND METHODS: CELLS and GEL
Cell Culture
-Embryonic forebrain removed + digested (rat)
-Single cells cultured on:
1) poly ornithine coated cover slips + media (control)
2) 3-D hydrogel construct
Hydrogel Preparation
-Components
PEG-Macromer (10wt%)
cells + media
photoinitiator (0.05wt%)
-Exposed to UV light 10 min
-Gel massed when dry/wet, calculate compressive modulus over time
(degradation rate)
MATERIALS AND METHODS: STAINING
Confocal Imaging:
Gels vibrotome sectioned
a) Stained for live/dead
 VIABILITY
Calcein-AM
Calcein-AM = membrane permeable
Cleaved calcein fluoresces, membrane impermeable
Ethidium bromide
Fluoresces red after binding DNA
 FUNCTION
b) Stained for calcium
Fluo-3 = calcium indicator
GABA applied to cells
Laser excited Fluo-3 measures calcium (GABA response)
MATERIALS AND METHODS: DNA, ATP
Obtaining cytosolic material
Hydrogel homogenized with lysis buffer
disrupts polymer gel
Biochemical Analysis
a) DNA content quantified with PicoGreen Assay
b) Protein content analyzed with Western Blot
- glial fibrillary acidic protein (GFAP)
- beta tubulin III
c) Immunocytochemical identification with antibodies (directly on gel)
- GFAP
- beta tubulin III
- nestin
- fibronectin
- synaptophysin
DOES HYDROGEL RESTRICT CELL GROWTH?
Differences between monolayer (plate) and hydrogel cultures
- 2-D v 3-D access to nutrients
- physical obstruction in 3-D gel
Is the hydrogel a physical barrier to growth?
Are nutrients directed toward replication or creating space?
1 week
Study: Culture both monolayer and 3-D hydrogel cultures
Measure DNA content (reflects population size)
Observations:
No statistical difference in DNA content between both
culture types (with p-value 0.05)
Conclusion:
Cells proliferate in early hydrogel equally well
DOES HYDROGEL AFFECT VIABILITY?
Study: Culture both monolayer and 3-D hydrogel cultures
Measure DNA, ATP content (reflects population size)
Observations:
DEAD CELLS
1 pg ATP
8230 pg DNA
6 pg ATP / pg DNA
Conclusion:
MONOLAYER (24h)
HYDROGEL (24h)
1486 ng ATP
7386 pg DNA
143 pg ATP / pg DNA
190 pg ATP / pg DNA
Hydrogel viability significantly greater as measured by
DNA content
DOES HYDROGEL AFFECT VIABILITY?
Column 1: healthy monolayer reference
201 pg ATP / pg DNA
Column 2: dead cells
Column 3: monolayer culture (24h)
143 pg ATP/ pg DNA
Column 4: hydrogel culture (24h)
190 pg ATP/ pg DNA
Column 5: hydrogel culture (16d)
215 pg ATP / pg DNA
Questions: How old is monolayer reference?
Day 16 monolayer ATP/DNA ratio?
CELL AGGREGATION IN HYDROGEL (DAYS <12)
Day 0: single cells distributed uniformly throughout gel
Day 3: single cells and cell
clusters (~ 20 um)
Day 7: cell clusters (~ 30 um)
Actively dividing
CELL AGGREGATION IN HYDROGEL
Mesh size increases 3x days 10-12
Gel completely hydrolyzed day 16
TISSUE FORMATION (DAYS > 12)
Plexus formation:
Days 10, 12, 14, 16
Temporal control
achieved with
different polymers
CELL / TISSUE MORPHOLOGY IN HYDROGEL
- Initial growth is proliferation, not
migration, based ~ hence clusters
- First 12 days (Fig. 3b)
Processes start to form
Processes wrap around cluster core
(not penetrate hydrogel)
Core ~ 17+/-4 um thick
- Days 13-14 (Fig. 3d)
Processes grow radially
Rapid hydrolysis of hydrogel
Mesh size increases 3x
Process length ~52um into hydrogel
CELL / TISSUE MORPHOLOGY IN HYDROGEL
Fig. 1b
- Mesh size inversely
proportional to modulus
- Processes penetrate hydrogel
at 2 wks
- PEG 2.5 glycolide decreases
time to 1 wk
- PEG 2 lactide increases time
to 3 wks
Possible to achieve temporal
control of tissue growth in 3-D
ECM IN HYDROGEL
During development:
Neurite receptors bind ECM
ECM provides traction force for neurite extension
ECM molecules
- Laminin
- Fibronectin
- Collagen IV
Normal Neurite ECM
Y
Y
Y
Hydrogel ECM
N
Y
N
CELL DIFFERENTIATION IN HYDROGEL
- Neural precursors forms neurons or glia
- Beta tubulin III  neurons
- GFAP  glia
Immunocytochemistry staining of hydrogel sections revealed:
- Day 0 ~ 2.6*10^6 cells
66% cells beta tubulin III positive
No GFAP
- Day 16 ~ 7.3*10^6 cells (3.5x increase)
35% cells beta tubulin III positive (Fig. 4b)
38% cells GFAP positive (Fig. 4a)
CELL DIFFERENTIATION IN HYDROGEL
- Neural precursors forms neurons or glia
- Beta tubulin III  neurons
- GFAP  glia
Immunocytochemistry staining
Western Blot
CELL FUNCTION IN HYDROGEL
Method: observe cellular response to GABA to assay functionality
1) Fluo-3 tags calcium within cells
2) GABA transmitter applied to cells
3) Response  cellular calcium influx  visible with Fluo-3
Observations:
Cells functional
Fast and slow response types
Did not mention
proportion functional
Typical calcium response
SUMMARY
- Achieved temporal control over
gel degradation
tissue formation
- NPC generates limited (but sufficient) ECM
- NPC proliferates AND differentiates
- 3-D scaffold not physical obstacle
- Proliferation/viability better than 2-D culture
CELL / TISSUE MORPHOLOGY IN HYDROGEL
Choice of graph?
Does this mean at day 7:
~ 60% cells at 30um clusters
and
~ 80% cells at 45um clusters?
Or at day 16:
~ 20% cells at 20um clusters
and
~ 80% cells at 70um clusters
?